Laboratory-based experimental research into the effect of diagenesis on heated bone: implications and improved tools for the characterisation of ancient fire

The use of fire is considered to be one of the most important cultural innovations in human evolution. Understanding the taphonomy of fire remains is an important prerequisite for valid interpretations of hominin fire-related behaviour. Presented here are the results of a series of laboratory-based experiments testing the effect of different pH conditions (acidic, neutral, alkaline) on the physical and chemical properties of heated bone (charred and combusted). By taking a fundamental-research approach the study gives insight into the specific effect of pH exposure and its underlying chemical processes, and provides data that can be applied to heated bone from any context and time period. Results show that diagenesis has a significant impact on the preservation potential of heated bones, as well as on the reliability of the analytical techniques used to reconstruct past heating conditions. The study provides reference data and a toolkit for the analysis of heated bone, that explicitly takes diagenesis into account, and in doing so offers a significant improvement to the accuracy with which we can reconstruct heating conditions and fire-related human behaviour in the past.


SI 1.7 Structural analysis SI 1.7.1 X-ray diffraction (XRD)
Single measurements were taken on fixed, powdered samples, placed in PMMA sample holders with a diameter of 25 mm and a depth of 1 mm. Analyses were carried out using a Bruker-AXS D8 advance powder x-ray diffractometer configured with LYNXEYE detector and DAVINCI design. The machine is equipped with two circle goniometer theta/2theta, and a ceramic 2.2 kW Cu K 1,2 tube (12 mm  = 1.54060 Å), and is operated at 40 kV and 40 mA. Measurements were collected in the 16-68 2 range, with a step size of 0.04 2, a step time of 1 sec, and 17 mm illumination. The gonio radius was 280 mm, and a 2.5 soller slit was used. The Crystallinity Index (CI) was calculated following Person et al. [SI.4]. Table SI.1: Overview of the samples used for the preservation experiments. pH 3 and pH 12 experiments were carried out in triplicate, single samples of selected temperatures were incubated in pH 7 solutions as control samples.
Figure SI.1: The custom oven rig used with the Carbolite GVA 12/300 tube furnace to produce the combusted bone samples. The rig allowed the samples to be placed in the middle of the furnace tube. The mesh platform allowed for sufficient airflow, while additional insulation at the top kept the temperature in the furnace more constant despite the airflow.  Table SI.2a, b, and c. While Munsell codes are a good way to describe the colour of heated bones in a systematic and reproducible way, the Munsell colour chart turned out not to adequately capture the colour variation of heated bone exposed to pH solutions. This was the case for the unheated samples, as well as those charred to 400 and 700 C and samples combusted at 600 C.
Exposure to pH 3 solutions results in darker colours. This is the case for bone charred to 300 and 400 C. At higher temperatures no colour change can be detected because all samples are black. For combusted bone exposure to pH 3 results in darker colours for samples heated to 200 -600 C. At 700 -900 C no colour change can be detected because all samples are stark white. The colour of unheated bone appears not to be affected by exposure to pH 3 conditions.
Exposure to pH 12 solutions results in lighter colours in both charred and combusted bone, as well as in unheated bone. Charred bone samples display a lighter colour at temperatures from 200 to 700 C, while for combusted bone lighter colours appear from 200 to 500 C.
Exposure to pH 7 solutions has no effect on the two available combusted bone samples (300 and 700 C), while it results in lighter colours in the two available charred bone samples and unheated bone sample (20, 300, 700 C).

SI 2.1.2 Fragmentation
Fragmentation as a result of exposure to different pH conditions occurred for 52% (n = 56) of the samples (n = 107), and in equal proportions for charred and combusted bone (Table SI. 3a, b, c). For both charred and combusted bone (both n = 50) 56% of the samples were fragmented after incubation. Notably, no fragmentation occurred for the unheated bone samples (n = 7). Exposure to pH 3 and pH 12 both resulted in 53% (n = 27) fragmentation of the samples (n = 51). The effect of pH 3 was slightly larger for combusted bone than for charred bone (58% vs. 54%), with the reverse being true for exposure to pH 12 (charred 58% vs. combusted 54%). Of the samples exposed to pH 7 (n= 5) 40% were fragmented (n = 2).
For the different temperature groups the data show that the percentage of fragmentation increases with increasing temperature (200-400 C: 32%; 500-600 C: 42%; 700-900 C: 90%). At 200-400 C combusted bone is more susceptible to fragmentation than charred bone, particularly when exposed to pH 3 (44% vs. 22%). However, at 500-600 C, combusted bone is much less susceptible to fragmentation than charred bone, regardless of pH (17% vs. 67%). At 700-900 C, 100% of the combusted bone samples are fragmented, versus 78% of the charred bone samples. For exposure to pH 7 only samples heated to 300 and 700 C were tested (n = 4), and among those only those heated to 700 C showed fragmentation (100%).
Fragmentation was also classified into groups based on number of fragments: 2-4, 5-7, and 8-10 pieces. In the first group (2-4 pieces) more fragments of charred bone are present than for combusted bone. For group two (5-7 pieces) the same number of fragments for charred and combusted bone is found, with the exception of combusted bone exposed to pH 12, which has double the number of fragments (17% vs 8%). In the third group (8-10 pieces) combusted bone samples prevail over charred bone samples, regardless of pH.

SI 2.1.3 Mass loss
Percentages of mass loss can be found in Fig. SI.3a and b, and in Table SI.2a, b, and c. Results show that exposure to acidic conditions of unheated bone results in much higher mass loss than exposure to neutral and alkaline conditions (15.4% vs. 5.6 and 5.7%).
The highest mass loss for exposure to pH 3 occurs for bones heated to 200 C (charred: 16.8%, combusted: 19.8%). At 300 C mass loss is still quite high (charred: 14.9%, combusted: 8.6%). At these temperatures the mass loss is lower than and similar to pH 12 values, respectively, for charred bone, while for combusted bone mass loss for pH 3 is higher for both temperatures. Between 400 and 900 C mass loss for charred bone exposed to pH 3 is consistently higher than for pH 12, and decreases with increasing temperature (from 9 to 5.4%). Mass loss for combusted bone at these temperatures is also consistently higher for pH 3 than for pH 12, but values fluctuate (between 8.2 and 4.6%).
The highest mass loss for exposure to pH 12 also occurs for bones heated to 200 C (charred: 20.9%, combusted: 18%), with the mass loss for charred bone exceeding those resulting from pH 3, and the values for combusted bone being slightly lower than those resulting from pH 3. At 300 C mass loss for charred bone is 15.1% (similar to pH 3) and 7.1% for combusted bone (<pH 3). From 400 C onwards mass loss values are very low (< 3%) and often dip below zero, suggesting a slight mass increase. The latter occurs more often for charred bone than for combusted bone (at 500-900 C vs. at 400 + 600 C).
At 300 C, mass loss for bone exposed to pH 7 is much lower than for that exposed to pH 3 and pH 12, both for charred and combusted bone (charred: 7.5%, combusted: 2.7%). At 700 C the effect of pH 7 is similar to that of pH 12 (charred: -5.3%, combusted: 1%).  Table SI.2a, b, and c. The effect of the different pH values will be discussed using the values for the organic content. However, it should be noted that any changes in the organic content will result in changes in the relative inorganic content, and vice versa. For example, a relative increase in organic content suggests loss of inorganic compounds. The data shows that exposure of unheated bone to acidic conditions results in a relative increase in organic content, in relation to unexposed bone (25.5 wt% vs. 24.1 wt%). Exposure to alkaline conditions results in a decrease in the organic content compared to unexposed bone (22.3 wt% vs. 24.1 wt%). Unheated bone exposed to neutral conditions appears to behave like unexposed bone (organic content: 23.8 wt%).
Exposure to pH 3 has the most pronounced effect on bones heated to 200 C, resulting in a decrease in organic content compared to unexposed bone at the same temperature (charred: 14.3 wt% vs. 24 wt%, combusted: 12.3 wt% vs. 24.9 wt%). At 300 C there is also a decrease in organic content compared to unexposed bone (charred: 10.4 wt% vs. 18.7 wt%, combusted: 12 wt% vs. 15 wt%). For bone charred to 400 C there is still a small decrease in organic content, while for combusted bone there is only a small relative increase in organic content compared to unexposed bone at the same temperature. At 500 C the organic content of charred bone exposed to pH 3 overlaps with that of unexposed bone. From 600 C onwards there is a relative increase in organic content, which peaks at 900 C. For combusted bone exposed to pH 3 there is a small relative increase in organic content for bone heated to 500-600 C, and values overlap with those of unexposed bone for temperatures of 700-900 C.
Exposure to pH 12 has the most pronounced effect on bones heated to 200 C, even more so than pH 3, resulting in a decrease in organic content compared to unexposed bone (charred: 3.3 wt% vs. 24 wt%, combusted: 2.7 wt% vs. 24.9 wt%). At 300 C the organic content of charred bone is similarly low, while for combusted bone the effect is slightly less intense (8.2 wt%). From 400-600 C there is still a decrease in organic content for charred bone, but the offset from unexposed bone is now much smaller. For combusted bone exposed to pH 12 the organic content overlaps with that of unexposed bone at temperatures of 400-700 C, with the exception of 600 C where there is a relative increase. For charred bone organic content values start to overlap with those of unexposed bone at 700-800 C. At the far end of the temperature range both charred and combusted bone samples show a minor relative increase in organic content, compared to unexposed bone of the same temperature. Charred bone exposed to pH 7 appears to behave like charred bone exposed to pH 3. Combusted bone heated to 300 C shows a decrease in organic content, but much less pronounced than in combusted bone exposed to pH 3 and pH 12. At 700 C all values overlap, regardless of pH. For all combinations of variables (charred, combusted, pH) the effect of pH exposure decreases with increasing temperature.

SI 2.2 Elemental analysis SI 2.2.1 X-ray fluorescence (XRF)
The results of the XRF analysis are presented in Fig. SI.5a, b, c, d, and Table SI.2a, b, and c. The effect of pH exposure on the elemental composition of heated bone is described based on the two main elements present in bone mineral: calcium and phosphorous. It should be noted that a comparison with the unexposed reference data could not be made. Therefore, only the pH samples will be evaluated here. The used XRF technique compensates elements it cannot detect (especially C, H, N, O) by a balance parameter. This means that when material is lost there is a relative increase in the remaining components. XRF results are therefore also affected by dissolution of compounds that cannot be directly measured by the technique itself, such as organic material (CH2O) and carbonate (CO3). The effect of exposure to pH solutions on the CaO and P2O5 content cannot be properly assessed without a comparison with unexposed reference data. However, there are clear indications of loss of organic material and carbonates in the XRF results.
For charred bone, exposure to pH 3 conditions results in lower CaO values than for samples exposed to pH 12 solutions. This effect is strongest at low temperatures (200 -300 C) and decreases with increasing temperature as the relative organic fraction increases as the inorganic components are lost. Combusted bone exposed to pH 3 conditions shows a similar trend at low temperatures (200 -400 C), after which CaO values start to overlap with those for samples exposed to pH 12 conditions. P2O5 values in low temperature charred bone (200 -300 C) exposed to pH 3 conditions are also lower than in bone exposed to pH 12 conditions. Values overlap at 400 C and become higher for pH 3 samples than for pH 12 samples from 500 C onwards. Combusted bone exposed to pH 3 conditions also has lower P2O5 values at 200 C. Values overlap with those for pH 12 samples at 300 C, and are higher at 400 to 700 C. At 800 and 900 C values for both pH conditions overlap and are very close together.
The data show that CaO and P2O5 values are higher for pH 12 samples than for pH 3 samples at low temperatures. In the samples exposed to pH3, there is still a lot of organic material present that would be lost during exposure to pH 12 conditions. CaO and P2O5 are higher in pH 3 samples than in pH 12 samples at higher temperatures (>500 C) where there is little to no effect of loss of organic content. This relative increase compared to the pH 12 treated samples might be related to loss of carbonates as a result of exposure to pH 3 conditions, as carbonate (CO3) is only lost at > 700 C in samples exposed to pH 12. Finally, overlapping values for both pH values indicate similar chemical properties (i.e., mainly bone mineral at high temperatures), as well as no or only a minor effect of pH exposure.

SI 2.3 Molecular analysis SI 2.3.1 Fourier transform infrared spectroscopy (FTIR)
The results of the FTIR analysis are presented in Table SI Exposure to pH 3 solutions mainly affects the inorganic content of both charred and combusted bone, resulting in reduced CO3 peaks and increased splitting of the PO4 3-v1 symmetric stretching. In charred bone the shoulder on the PO4 3-v3 peak appears at a much lower temperature (at 400 C instead of at 900 C). The changes to the PO4 3peaks suggest both recrystallisation and some loss of phosphates. Loss of organics can also be observed (amides and aromatic compounds), but to a lesser extent than for samples exposed to pH 12 solutions. The loss of CO3 also becomes clear from the C/P ratio values, which are always lower for bone exposed to pH 3 solutions than for unexposed bone. The C/P ratio differences between pH 3 and pH 12 samples decrease with increased temperature, with the ratio difference being largest for bone heated to 200 C. Exposure to pH 3 conditions also has a clear effect on the splitting factor, with higher values compared to unexposed bone. The effect is always more pronounced for bone exposed to pH 3 than for those exposed to pH 12, and for charred bone the difference between the two increases with higher temperatures (400 -900 C).
Exposure to pH 12 solutions mainly affects the organic content of charred and combusted bone, resulting in loss of amides and aromatic compounds. Some changes in the inorganic content can also be observed (e.g., reduction of CO3 peaks and increased splitting of the PO4 3-v1 symmetric stretching), but the effect is less pronounced than for samples exposed to pH 3 solutions. The reduction in CO3 can also be tracked through the C/P ratio. For bone exposed to pH 12 conditions C/P values are lower than those of unexposed bone for all temperatures, except at 600 -800 C for charred bone and at 500 -700 C for combusted bone. C/P values for bone exposed to pH 12 are always higher than those for bone exposed to pH 3. It should be noted that the reduction of CO 3 peaks likely relates both to loss of carbonates and to loss of organic compounds, with which these peaks overlap. There is also an increase in crystallinity, as reflected by the higher SF values as compared to unexposed heated bone. However, this effect is not as strong as for exposure to pH 3 solutions.
Exposure to pH 7 solutions also results in a decrease in organic compounds and CO3, as well as increased splitting of the PO4 3symmetric stretching and increased SF values. However, the effect is generally less pronounced than for the other two pH values.
The results of the Py-GCMS analysis are grouped in eight different compound types and presented in Table SI.4a, b, and c. The identified compound types are monocyclic aromatic hydrocarbons (MAH), methylene chain compounds (MCC), N-containing compounds (NCOMP), polycyclic aromatic hydrocarbons (PAH), phenols (PHEN), tert-butylphenols (TERT), triterpenoids (TRITERP), and unidentified (OTHER). Due to the difference in technique and resolution (Py-GCMS vs. pyMS and DTMS), a comparison with the heated bone MS reference data is not warranted. Py-GCMS results show that exposure to all pH values has an effect on the organic molecular composition of heated bone. Unheated bone is also affected by pH exposure, but to a lesser extent than heated bone.
Exposure to pH 3 solutions results in dissolution and loss of MAHs (e.g., benzene, toluene, styrene) for both charred and combusted bone, and for all analysed temperatures (20 C, 200-500 C). The effect is most noticeable for bone heated to 200 C and decreases with increasing temperature. MCCs (e.g., alkyl-nitrile, amide, alkanes) and PAHs (e.g., indene, naphthalene, fluorene) are affected by exposure to pH 3 in the low temperature samples, while they are affected by exposure to pH 12 in the medium temperature samples. Exposure to pH 12 solutions results in dissolution and loss of NCOMPs (e.g., pyrrole, pyridine, dipeptides), PHENs (e.g., phenol), TRITERPs (e.g., cholestadiene, cholestadienol), and some unidentified compounds (OTHER) for both charred and combusted bone, as well as for unheated bone. For NCOMPs, the largest category, many compounds are completely lost as a result of exposure to pH 12, especially for bone heated to 200 C (e.g., diketodipyrrole). The effect decreases with increasing temperature, alongside the overall decrease in NCOMPs as a result of heating. In both charred and combusted bone PHENs leach out as a result of exposure to pH 12 solutions, sometimes completely (e.g., at 200 C). TRITERPs, present at low temperatures (20 C, charred 200-300 C, combusted 200 C), are also completely lost after exposure to pH 12. While being affected by pH 3 at low temperatures, MCCs and PAHs leach out in pH 12 at higher temperatures (charred bone: MCC at 400-500 C, PAH at 500 C; combusted: MCC at 500 C, PAH at 400-500 C). Finally, TERTs leach out in pH 12 solutions for bone charred to 500 C and for bone combusted to 300 C.
For exposure to pH 7 solutions only 3 samples were available for Py-GCMS analysis: unheated bone, charred 300 C, and combusted 300 C. For unheated bone the effect of pH 7 is somewhere in between that of pH 3 and pH 12, i.e., some loss of compounds. However, at 300 C there is no clear pattern and sometimes compound values are lower after exposure to pH 7 than for exposure to the other two pH values.

SI 2.4 Structural analysis SI 2.4.1 X-ray diffraction (XRD)
The results of the XRD analysis are presented in Fig. SI.9a and  Exposure to pH 3 results in the largest increase in crystallinity, for all charred bone samples and for combusted bone heated between 500 and 600°C. In both cases the effect increases with increasing temperature. The effect is also visible in the diffractograms as sharper, more pronounced peaks over the full 2 range for samples exposed to pH 3. The exception to this trend is the combusted bone samples heated to 700°C and above. For these samples there is still an increase in crystallinity, but here the effect of pH 12 is more pronounced. Exposure to both pH values results in a decrease in crystallinity for combusted bone heated to 900°C. This may be related to the process of dehydroxylation of the bone mineral, which is known to start around that temperature in combusted bone (Tonsuaadu et al., 2012).
Exposure to pH 12 results in an increase in crystallinity in charred bone that is less severe compared to exposure to pH 3. For 600 and 700°C CI values are higher, but still very similar to those of unexposed charred bone. The increase in crystallinity becomes a bit bigger for bone charred to 800 and 900°C. For combusted bone heated to 500°C and exposed to pH 12 the CI values overlap with those of samples exposed to pH 3, but are much higher than those of unexposed bone. For combusted bone heated to 600°C the increase in crystallinity is similar, but lower than for the samples exposed to pH 3. At 700 and 800°C, the trend reverses and samples exposed to pH 12 start showing the highest crystallinity increase. At 900°C another switch occurs, now resulting in a decrease in crystallinity, which is less defined for pH 12 than for pH 3. This may be related to dehydroxylation reactions that are known to occur in combusted bone at this temperature [SI.6].
Exposure to pH 7 appears most similar to exposure to pH 12. However, it should be noted that only two pH 7 samples were available for XRD analysis (charred + combusted bone 700°C).  Figure SI.2a,b,c: Overview of the variation in colour for charred (A) and combusted (B) bone exposed to pH 3 and pH 12 conditions, as well as control samples exposed to pH 7 conditions (C). SI.2a and b are duplicates of Fig. 1a and b presented in the main text.
A°C B°C 21 C Combusted Charred°C Figure SI.4: Graphs showing the variation in organic content (wt%) in charred (A) and combusted (B) bone, as measured by TGA, as a result of pH exposure. Triangles = unheated bone, circles = heated bone, grey = untreated samples, yellow = pH 3 samples, blue = pH 7 samples, green = pH 12 samples. These graphs are duplicates of Fig. 5a and b presented in the main text. 28 Figure SI.8a-q: FTIR spectra for all temperature-pH combinations, compared to their untreated equivalent. Unheated (A) above, charred and combusted bone (B-Q) below, with charred bone in the left column and combusted bone in the right column. Grey = untreated, yellow = pH 3, blue = pH 7, green = pH 12. For peak identifications see Fig.  4 in the main text. SI.8d, e, l, and m are duplicates of Fig. 4a, b, and c presented in the main text.
Figure SI.10a-k: XRD diffractograms for all temperature-pH combinations, compared to their untreated equivalent. Unheated (A) above, charred and combusted bone (B-K) below, with charred bone in the left column and combusted bone in the right column. Grey = untreated, yellow = pH 3, blue = pH 7, green = pH 12. For peak identifications see Fig. 3 in the main text. SI.10f and g are duplicates of Fig. 3a and b